This invention relates to a method and material for enhancing gene expression in organisms, particularly in plants. One particular, but not exclusive, application of the invention is the enhancement of caroteniod biosynthesis in plants such as tomato (Lycopersicon spp.)
In order to increase production of a protein by an organism, it is known practice to insert into the genome of the target organism one or more additional copies of the protein-encoding gene by genetic transformation. Such copies would normally be identical to a gene which is already present in the plant or, alternatively, they may be identical copies of a foreign gene. In theory, multiple gene copies should, on expression cause the organism to produce the selected protein in greater than normal amounts, this is referred to as "overexpression". Experiments have shown however, that low expression or no expression of certain genes can result when multiple copies of the gene are present. (Napoli et al 1990 and Dorlhac de Borne et al 1994). This phenomenon is referred to as co-suppression. It most frequently occurs when recombinant genes are introduced into a plant already containing a gene similar in nucleotide sequence. It has also been observed in endogenous plant genes and transposable elements. The effects of co-suppression are not always immediate and can be influenced by developmental and environmental factors in the primary transformants or in subsequent generations.
The general rule is to transform plants with a DNA sequence the codon usage of which approximates to the codon frequency used by the plant. Experimental analysis has shown that introducing a second copy of a gene identical in sequence to a gene already in the plant genome can result (in some instances) with the expression of the transgene, endogenous gene or both genes being inactivated (co-suppression). The mechanisms of exactly how co-suppression occurs are unclear, however there are several theories incorporating both pre- and post-gene transcriptional blocks.
As a rule the nucleotide sequence of an inserted gene is "optimised" in two respects. The codon usage of the inserted gene is modified to approximate to the preferred codon usage of the species into which the gene is to be inserted. Inserted genes may also be optimised in respect of the nucleotide usage with the aim of approximating the purine to pyrimidine ratio to that commonly found in the target species. When genes of bacterial origin are transferred to plants, for example, it is well known that the nucleotide usage has to be altered to avoid highly adenylated regions, common in bacterial genes, which may be misread by the eukaryotic expression machinery as a polyadenylation signal specifying termination of translation, resulting in truncation of the polypeptide. This is all common practice and is entirely logical that an inserted sequence should mimic the codon and nucleotide usage of the target organism for optimum expression.
An object of the present invention is to provide means by which co-suppression may be obviated or mitigated.